The effect of the regulatory proteins, troponin and tropomyosin, on the binding of myosin subfragment-one to actin was determined in the presence of different nucleotides. We found that in the presence of AMP-PNP, PPI, and ADP, tropomyosin alone confers slight positive cooperativity on the binding of S-1 to actin since there is a 3-fold increase in the affinity of S-1 for actin as the actin sites become saturated with S-1. The effect of tropomyosin on this binding is indistinguishable from that obtained with troponin.tropomyosin in the presence of Ca-2++. However, in the absence of Ca-++, troponin.tropomyosin causes pronounced cooperativity in the binding of S-1 to actin, i.e. when the binding sites on actin are mostly unoccupied, troponin.tropomyosin strongly inhibits this binding, whereas when most of the sites are occupied, it strengthens the binding. These results indicate that troponin affects the binding of S-1 to regulated actin only in the absence of Ca-++ and not in the presence of CA-++.